Objective: To explore if conventional protein kinase C (cPKC: PKCα and PKCβ) contributes to paraquat (PQ) -induced abnormal permeability of mouse brain microvascular endothelial cells (BMECs) via the regulation of tight junction (TJ) proteins. Methods: The immortalized mouse brain endothelial cell line (bEnd.3) was used to establish a monolayer blood-brain barrier (BBB) model. In order to evaluate the function of the in vitro BBB model, the transendothelial electrical resistance (TEER) and permeability were measured by a Millicell-ERS volt-ohmmeter and sodium fluorescent (Na-FLU) , respectively. MTT assay was used to determine the relative survival rate of cells. The dose-response relationship was determined by treating cells with 0, 50, 100, 200, and 300 μmol/L PQ for 24 hours. The time-response relationship was determined by treating cells with 200 μmol/L PQ for 6, 12, 24, 48, and 72 hours. After the treatment of cells with 0, 100, 200, and 300 μmol/L PQ for 24 hours, the protein and mRNA expression levels of ZO-1, Occludin, and Claudin-5 were measured by immunofluorescence (IF) and quantitative real-time RT-PCR (qRT-PCR) , respectively; the expression of PKCα, PKCβ, phosphorylated (p) -PKCα, and p-PKCβ was determined by Western blot. After the treatment of cells with 200? mol/L PQ for 24 hours following the pretreatment with a classical PKC inhibitor (Go 6983, 1 μmol/L) for 1 hour, the protein expression of ZO-1, Occludin, Claudin-5, p-PKCα, and p-PKCβ was measured by Western blot. Results: The TEER of the bEnd. 3 cells increased gradually with the cell culture time, and reached a peak value of 114.3±6.9 Ω·cm(2) on day 6. According to the permeability analysis by Na-FLU, cell permeability gradually decreased with the cell culture time, and reached 1.7±0.2 cm/min on day 6, suggesting a well-behaved barrier function of cells. Compared with the control group, the survival rates of the bEnd.3 cells were significantly reduced after exposure to 100, 200, or 300 μmol/L PQ for 24 hours (P<0.05) , or after exposure to 200 μmol/L PQ for 6, 12, 24, 48, or 72 hours (P <0.05) , indicating a dose-and time-dependent relationship. The IF and qRT-PCR results showed that the protein and mRNA expression levels of ZO-1, Occludin, and Claudin-5 were significantly reduced with the increase in the concentration of PQ (P<0.05) . The Western blot analysis showed that compared with the control group, cells exposed to PQ had significantly higher protein expression of p-PKCα and p-PKCβ and significantly lower protein expression of ZO-1, Occludin, and Claudin-5 (P<0.05) . Compared with the PQ treatment group, the Go 6983 intervention group had significantly higher protein expression of ZO-1, Occludin, and Claudin-5 and significantly lower protein expression of p-PKCα and p-PKCβ (P<0.05) . Conclusion: By activation of cPKC (PKCα and PKCβ) , PQ reduces the protein and mRNA expression of TJ proteins and enhances the permeability of murine BMECs.
目的： 探讨经典型蛋白激酶C（cPKC：PKC α、PKC β）是否通过调控紧密连接蛋白（tight Junction，TJ）的表达，引起百草枯（paraquat，PQ）致小鼠脑微血管内皮细胞通透性的异常。 方法： 体外培养小鼠脑组织来源的血管内皮细胞株（bEnd.3）作为单层血脑屏障模型，通过Millicell-ERS细胞电阻仪测定跨内皮细胞电阻（TEER）、荧光素钠（Na-FLU）通透性测定以评价体外血脑屏障模型的功能。四甲基偶氮唑蓝（MTT）法测定细胞相对存活率：终浓度0、50、100、200、300 μmol/L PQ处理细胞24 h，确定剂量-效应关系；终浓度200 μmol/L PQ分别处理细胞6、12、24、48、72 h，确定时间-效应关系。终浓度0、100、200、300 μmol/L PQ处理细胞24 h，免疫荧光（IF）和实时荧光定量PCR（qRT-PCR）分别测定紧密连接蛋白（ZO-1、Occludin、Claudin-5）和基因的表达水平，免疫印迹法（Western blot）测定PKC α、PKC β、p-PKC α、p-PKC β蛋白表达水平；经典PKC抑制剂（Go 6983）1 μmol/L预处理细胞1 h后，200 μmol/L PQ染毒细胞24 h，Western blot法测定ZO-1、Occludin、Claudin-5及p-PKC α、p-PKC β蛋白表达水平。 结果： bEnd. 3细胞的TEER随培养时间延长逐渐升高，于第6 d达到峰值[（114.3±6.9）Ω· cm(2)]，荧光素钠通透性检测显示，细胞通透性随培养时间延长逐渐降低，于第6天降至（1.7±0.2）cm/min，说明细胞屏障功能良好。与对照组比较，100、200、300 μmol/L PQ染毒组细胞存活率明显降低，200 μmol/L PQ染毒细胞12、24、48、72 h，细胞存活率明显降低，差异有统计学意义（P<0.05），呈剂量依赖和时间依赖关系。IF和qRT-PCR显示，随着PQ浓度的升高，ZO-1、Occludin、Claudin-5蛋白和基因表达量明显减少，差异均有统计学意义（P<0.05）。Western blot法检测显示，与对照组比较，PQ染毒组的p-PKC α、p-PKC β蛋白表达水平明显升高，ZO-1、Occludin、Claudin-5蛋白表达量明显降低，差异均有统计学意义（P<0.05）；Go 6983干预组的ZO-1、Occludin、Claudin-5蛋白表达水平较染毒组明显升高，p-PKC α、p-PKC β蛋白表达水平明显下降，差异均有统计学意义（P<0.05）。 结论： PQ通过激活cPKC（PKC α、PKC β）导致紧密连接蛋白/基因表达降低，引起小鼠脑微血管内皮细胞通透性升高。.
Keywords: Blood-brain barrier; Brain microvascular endothelial cells; Paraquat; Protein kinase C; Tight junction.